Abstract
Azathioprine (AZA) is widely used in clinical practice for preventing graft rejection in organ transplantations and various autoimmune and dermatological diseases with documented unpredictable hepatotoxicity. The potential molecular cytotoxic mechanisms of AZA towards isolated rat hepatocytes were investigated in this study using “Accelerated Cytotoxicity Mechanism Screening” techniques. The concentration of AZA required to cause 50% cytotoxicity in 2 hrs at 37°C was found to be 400 μM. A significant increase in AZA-induced cytotoxicity and reactive oxygen species (ROS) formation was observed when glutathione- (GSH-) depleted hepatocytes were used. The addition of N-acetylcysteine decreased cytotoxicity and ROS formation. Xanthine oxidase inhibition by allopurinol decreased AZA-induced cytotoxicity, ROS, and hydrogen peroxide (H2O2) formation and increased % mitochondrial membrane potential (MMP). Addition of N-acetylcysteine and allopurinol together caused nearly complete cytoprotection against AZA-induced hepatocyte death. TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl), a known ROS scavenger and a superoxide dismutase mimic, and antioxidants, like DPPD (N,N′-diphenyl-p-phenylenediamine), Trolox (a water soluble vitamin E analogue), and mesna (2-mercaptoethanesulfonate), also decreased hepatocyte death and ROS formation. Results from this study suggest that AZA-induced cytotoxicity in isolated rat hepatocytes may be partly due to ROS formation and GSH depletion that resulted in oxidative stress and mitochondrial injury.
Highlights
Azathioprine (AZA), prodrug of 6-mercaptopurine, is widely used as an immunosuppressant for several diseases such as inflammatory bowel disease (IBD) and autoimmune diseases and following transplantation to avoid organ rejection [1,2,3,4]
We investigated different toxicity routes of AZA towards isolated rat hepatocytes using Accelerated Cytotoxicity Mechanism Screening” (ACMS) techniques
A concentration and time dependent increase in cytotoxicity and reactive oxygen species (ROS) formation and a decrease in % membrane potential (MMP) were observed with AZA (100–500 μM) compared to control hepatocytes (Figure 1) incubated for 3 hrs
Summary
Azathioprine (AZA), prodrug of 6-mercaptopurine, is widely used as an immunosuppressant for several diseases such as inflammatory bowel disease (IBD) and autoimmune diseases and following transplantation to avoid organ rejection [1,2,3,4]. Previous studies performed with rat hepatocyte primary cultures showed that toxic concentrations of AZA (25–250 μM) led to profound intracellular GSH depletion, mitochondrial injury, metabolic activity reduction, decreased adenosine 5-triphosphate (ATP) levels, and cell death due to necrosis, not apoptosis. Similar effects were observed by Menor and colleagues [5] where AZA (150 μM) decreased the viability of rat hepatocytes and induced intracellular GSH depletion, metabolic activity reduction, and lactate dehydrogenase release.
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